The goal of this project is to definitively assess the role of genes identified in Project 1 and 2 in VCFS etiology. We plan to accomplish this goal by generating mice with mutations in four genes that are within or close to the minimally deleted region of chromosome 22. This phase will be followed by a set of experiments which will give us two strains of mice, each of which carries a deletion covering part of the VCFS critical region. Markers from the 500 kb minimally deleted region will be used to obtain yeast artificial chromosomes (YACs) containing the corresponding mouse Dna on chromosome 16. The YACs will be used to generate a sequence tagged site (STS)-content map. The contig will be oriented on chromosome 16 and the transcriptional orientation of three genes, 3911b2, IDD and ARV, with respect to MMU16 centromere and telomere will be established. It is anticipated that the order of these genes of MMU16 will be cen- ARV-IDd-39lb2-tel. We will use a three step procedure to generate deletions. The first step involves introduction of a lox P site containing selectable gene cassette into the IDD gene of ES cells. The second step involves modification of IDD mutation containing cells at the 391b2 and ARV loci separately. The third step is to transiently transfect a cre expression construct to generate IDD-391b2 and IDD-ARV deletions. We will use each of the different modified cells to generate mice. We propose to generate 3 knock-out mice (IDD, ARV, 391b2) and two deletion containing mice (IDD-391b2 and IDD-ARV). In addition, we will also construct mice carrying a mutation in a new clathrin heavy chain gene whose human counterpart is located in 22q11 and has high levels of expression in skeletal muscle. Each of these mice will be assessed for the phenotypes that might result from the gene targeted events. We will also assess if any of the knock-outs or deletions will yield developmental disorders akin to those seen in VCFS.